Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.

CV(%) = SD/mean x 100

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Cat.No E0604Po

Standard Curve Range: 2ng/ml - 600ng/ml

Sensitivity: 1.26ng/ml

Size: 96 wells

Storage: Store the reagents at 2-8°C. For over 6-month storage refer to the expiration date keep it at -20°C. Avoid repeated thaw cycles. If individual reagents are opened it is recommended that the kit be used within 1 month.

*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.

Intended Use

This sandwich kit is for the accurate quantitative detection of Porcine Syndecan-1 (also known as SDC1) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle

This ELISA Test Kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Porcine SDC1 antibody. SDC1 present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Porcine SDC1 Antibody is added and binds to SDC1 in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated SDC1 antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Porcine SDC1. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

Precautions

Prior to use, the ELISA Test Kit and sample should be warmed naturally to room temperature 30 minutes.

This instruction must be strictly followed in the experiment.

Once the desired number of strips has been removed, immediately reseal the bag to protect the remain from deterioration. Cover all reagents when not in use.

Make sure pipetting order and rate of addition from well-to-well when pipetting reagents.

Pipette tips and plate sealer in hand should be clean and disposable to avoid cross-contamination.

Avoid using the reagents from different batches together.

Substrate solution B is sensitive to light, don’t expose substrate solution B to light for a long time.

Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample matrix interference may falsely depress the specificity and accuracy of the assay.

Reagent Preparation

All reagents should be brought to room temperature before use.

Standard Reconstitute the 120μl of the standard (640ng/ml) with 120μl of standard diluent to generate a 320ng/ml standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (320ng/ml) 1:2 with standard diluent to produce 160ng/ml, 80ng/ml, 40ng/ml and 20ng/ml solutions. Standard diluent serves as the zero standard(0 ng/ml). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:

320ng/ml

Standard No.5

120μl Original Standard + 120μl Standard Diluent

160ng/ml

Standard No.4

120μl Standard No.5 + 120μl Standard Diluent

80ng/ml

Standard No.3

120μl Standard No.4 + 120μl Standard Diluent

40ng/ml

Standard No.2

120μl Standard No.3 + 120μl Standard Diluent

20ng/ml

Standard No.1

120μl Standard No.2 + 120μl Standard Diluent

Standard Concentration

Standard No.5

Standard No.4

Standard No.3

Standard No.2

Standard No.1

640ng/ml

320ng/ml

160ng/ml

80ng/ml

40ng/ml

20ng/ml

Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 600 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.

Summary

1. Prepare all reagents, samples and standards.

2. Add sample and ELISA reagent into each well. Incubate for 1 hour at 37°C.

3. Wash the plate 5 times.

4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.

5. Add stop solution and color develops.

6. Read the OD value within 10 minutes.

Calculationof Result

Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.